Cells function in the context of their environment. Cell surface receptors interact with extracellular components, initiating signaling pathways that regulate the organization of the cytoskeleton and gene expression. Generally, cellular interactions with the extracellular matrix are considered to promote adhesion. Adhesion is the process by which a cell attaches to a substrate and undergoes spreading with subsequent formation of stress fibers linked to the extracellular matrix through integrin containing focal adhesions. Extracellular factors also exist that regulate cell function by stimulating reversal of one or more of the stages of the adhesion process. PDGF activation of phosphoinositol (PI) 3-kinase and production of phosphatidylinositol (3,4,5)-trisphosphate [PtdIns (3,4,5)-P3] modulates the adhesive interaction between rat embryonic fibroblasts and the extracellular matrix. A-actinin is a potential target for the second messenger action of PtdIns (3,4,5)-P3. This proposal focuses on the mechanisms by which PDGF regulates the structure of focal adhesion plaques and the adhesive strength of the cell. Specific aim 1 is to characterize the binding of phosphoinositides to a-actinin. Radiolabeling, mass spectrometry, antiphosphoinositide antibodies, and fluorescent phosphoinositides will be used to quantitate phosphoinositide binding to a-actinin and determine cellular localization. Specific aim 2 will test the hypothesis that phosphoinositides differentially regulate the structure and function of a-actinin. Site-directed mutagenesis of the phosphoinositide binding site and x-ray crystallography will be used to accomplish this aim. Specific aim 3 is to test the hypothesis that phosphoinositide binding to a-actinin regulates cell adhesion and motility. These studies will involve adhesion, motility, adhesive strength, and contractility assays. Cell adhesion and growth factors are potential targets for xenobiotics. Knowledge of the mechanisms involved in the regulation of these processes is important for understanding the effects of environmental toxins on human health.